CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

Abstract

A unique feature of CD40 among the TNF receptor (TNFR) superfamily is its exquisitely contextual effects, as originally demonstrated in normal and malignant B-lymphocytes. We studied renal cell carcinoma (RCC) in comparison to normal (human renal proximal tubule) cells, as a model to better understand the role of CD40 in epithelial cells. CD40 ligation by membrane-presented CD40 ligand (mCD40L), but not soluble CD40 agonist, induced extensive apoptosis in RCC cells; by contrast, normal cells were totally refractory to mCD40L. These findings underline the importance of CD40 ‘signal-quality’ on cell fate and explain the lack of pro-apoptotic effects in RCC cells previously, while confirming the tumour specificity of CD40 in epithelial cells. mCD40L differentially regulated TRAF expression, causing sustained TRAF2/TRAF3 induction in RCC cells, yet downregulation of TRAF2 and no TRAF3 induction in normal cells, observations strikingly reminiscent of TRAF modulation in B-lymphocytes. mCD40L triggered reactive oxygen species (ROS) production, critical in apoptosis, and NADPH oxidase (Nox)-subunit p40phox phosphorylation, with Nox blockade abrogating apoptosis thus implying Nox-dependent initial ROS release. mCD40L mediated downregulation of Thioredoxin-1 (Trx-1), ASK1 phosphorylation, and JNK and p38 activation. Although both JNK/p38 were essential in apoptosis, p38 activation was JNK-dependent, which is the first report of such temporally defined JNK-p38 interplay during an apoptotic programme. CD40-killing entrained Bak/Bax induction, controlled by JNK/p38, and caspase-9-dependent mitochondrial apoptosis, accompanied by pro-inflammatory cytokine secretion, the repertoire of which also depended on CD40 signal quality. Previous reports suggested that, despite the ability of soluble CD40 agonist to reduce RCC tumour size in vivo via immunocyte activation, RCC could be targeted more effectively by combining CD40-mediated immune activation with direct tumour CD40 signalling. Since mCD40L represents a potent tumour cell-specific killing signal, our work not only offers insights into CD40’s biology in normal and malignant epithelial cells, but also provides an avenue for a ‘double-hit’ approach for inflammatory, tumour cell-specific CD40-based therapy.

Novel induction of CD40 expression by tumor cells with RAS/RAF/PI3K pathway inhibition augments response to checkpoint blockade

“…Notably, when RGS was combined with any of these RAS pathway inhibitors, the induction of CD40 expression in melanoma cells was further increased, suggesting pathways downstream of RAS may cooperate to restrain baseline CD40 expression in melanoma cells. It has been reported that CD40 signaling induces malignant cell death in many cancer types, including renal cell carcinoma [ 29 ], urothelial cell carcinoma [ 30 ], ovarian carcinoma [ 31 , 32 ], cervical carcinoma [ 33 ] and bladder carcinoma [ 34 ]. Here, we identified that the death rate of CD40 + melanoma cells was 3-fold higher than in CD40 − melanoma cells for both RGS-treated YUMM3.3 (Fig.…”

“…Proinflammatory cytokines, such as interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα), and epigenetic modulators, such as histone deacetylase (HDAC) inhibitors, can induce CD40 expression on melanoma cells [ 27 , 28 ]. Importantly, the induction of CD40 on cancer cells, but not normal cells, promotes apoptotic and/or necrotic signaling, which results in the cell death of renal cell carcinoma [ 29 ], urothelial cell carcinoma [ 30 ], ovarian carcinoma [ 31 , 32 ], cervical carcinoma [ 33 ] and bladder carcinoma [ 34 ]. Besides the direct cytotoxic effects in cancer cells, the ligation of CD40 on human melanoma cells also modulates tumor immunogenicity through upregulation of the expression of major histocompatibility complex (MHC) molecules and the production of proinflammatory factors (e.g., IL-6, IL-8 and TNFα, etc.)…”

METTL13 inhibits progression of clear cell renal cell carcinoma with repression on PI3K/AKT/mTOR/HIF-1α pathway and c-Myc expression

“…Furthermore, the database IntAct provided us with more than 20 proteins potentially combining with METTL13. Among these interactors, TRAF2 influences mitochondrial apoptosis of ccRCC [ 35 ]; KLK6’s expression is negatively associated with renal carcinoma grades [ 36 ]; higher HLA-E mRNA level predicts better prognosis of ccRCC patients [ 37 ]; FAS can be potentially regarded as a biomarker for predicting survival of renal cancer patients who have received nephrectomy [ 38 ]. The result also involved Myc, a family of proto-oncogenes, which extensively functions in cancer formation and development [ 39 ].…”

METTL13 Inhibits Progression of Clear Cell Renal Cell Carcinoma with Repression on PI3K/AKT/mTOR/HIF-1α Pathway and c-Myc Expression

“…The database IntAct, which aims at collecting data of protein interaction, provided us with more than 20 genes potentially combining with METTL13. Among these interactors, TRAF2 in uences mitochondrial apoptosis of ccRCC [38]; KLK6's expressions are negatively associated with renal carcinoma grades [39]; higher HLA-E mRNA level predicts better prognosis of ccRCC patients [40]; FAS can be regarded as a biomarker for predicting survival of renal cancer patients who have received nephrectomy [41]. The result also involved Myc, a family of transcriptional factors encoded by the proto-oncogene family, which extensively functions in cancer formation and development [42].…”